The objective of the proposed research is to understand the post-translational processing of 1- dystroglycan (1-DG), a glycoprotein that is a key component of the dystrophin-glycoprotein complex, anchoring muscle cells to the extracellular matrix. Defective glycosylation of 1-DG in its highly conserved central mucin-like region, with numerous glycosylated S and T residues is the cause of several forms of hereditary muscular dystrophy. Pendant glycans are linked to the protein by either N-acetylglucosamine (GalNAc) or the more unusual mannose (Man) residue whose sites are now just being mapped. Relationships between problems in assembly of the O-Man tetra-saccharide and disease have been established. We have recently found evidence that the GalNAc residues are important structurally. Synthetic, biochemical and structural methods will be integrated in the research to develop an understanding of the steps in this complex process, providing a quantitative description of enzymatic transformations and also facilitating insights into key interactions in which 1-DG participates. An understanding of the steps in this process will provide basis for rational design of therapies to correct the defects, including gene therapy and up-regulation of other enzymes that may complement the defective ones. The focus will be on two steps following the initial O-Man modifications of 1-DG. The two major aims are 1) to analyze the substrate profile of POMGnT1, an enzyme involved in a key step in the O-Man glycan assembly whose defects are associated with muscle-eye-brain disease, to better understand how rescue the defect and to develop a better assay for detection of defective enzyme, and 2) to elucidate the activity of polypeptide GalNAc transferases in initiating O-GalNAc sites on 1-DG and the relationship of these to POMGnT1 glycosylation. PUBLIC HEALTH RELEVANCE: Several forms of muscular dystrophy are associated with aberrations in the attachment of carbohydrates to the glycoprotein 1-dystroglycan arising from defects in the enzymes that carry out the complex series of steps leading to the specific installation of carbohydrates on it. The proposed research would use an integrated approach combining chemical synthesis of glycopeptides from1- dystroglycan, biochemical analysis of their modification by glycosyltransferase enzymes, and structural analysis of the reactants and products, to develop an understanding at a molecular level of the post-translational modification of this important glycoprotein. The information derived will contribute to understanding the pathological mechanisms of several forms of muscular dystrophies, improving clinical assay, and rational design of therapeutic interventions.